Determining the role of oxysterols in lymphocyte homing to lymph nodes in homeostasis and inflammation

PROJECT SUMMARY/ABSTRACT
Lymphocyte entry into lymph nodes (LNs) from blood is a key homeostatic process for efficient initiation of an
adaptive immune response to pathogens. Naïve B and T cells traffic through LNs to scan for foreign antigens
delivered to and concentrated in these organs from diverse sites of potential infection. If no antigens are
encountered, lymphocytes exit LNs into lymphatic circulation before returning to the blood and beginning the
cycle again. This constant lymphocyte recirculation requires large scale extravasation into lymphoid tissue under
non-inflammatory conditions, and the specialized vessels supporting this process in LNs are called high
endothelial venules (HEVs). HEVs express and luminally present several vascular ‘addressins’ and chemokines
that together support a multi-step adhesion cascade for lymphocyte entry into LNs. The signals promoting
lymphocyte arrest on HEV walls are well-elucidated, but the chemoattractants driving transmigration across the
endothelium are not fully understood. Elucidating the ligands and receptors that mediate this fundamental step
of entry is essential for understanding and manipulating immune cell trafficking in diseases such as cancer and
autoimmunity. The enzyme Ch25h, which produces 25-hydroxycholesterol (25HC) from cholesterol, is highly
and selectively expressed in HEVs compared to capillary endothelium in LNs. Furthermore, Cyp7b1, which
hydroxylates 25HC to generate 7𝛼𝛼,25-dihydroxycholesterol (7,25HC), is expressed in stromal cells surrounding
LN HEVs. 7,25HC is a potent ligand for EBI2, a GPCR that helps guide activated B cell movement within LNs
and is also highly expressed in naïve B and T cells. The capability for oxysterol synthesis by HEVs suggests
EBI2 and 7,25HC may play a role in mediating lymphocyte entry into LNs. We hypothesize that a gradient of
7,25HC across HEVs supports post-adhesion transendothelial migration of naïve B and T cells. In preliminary
studies, EBI2 KO B cells and CD4 T cells displayed a recruitment defect to LNs in adoptive transfers, and a
similar homing defect was evident in mice lacking Ch25h or Cyp7b1. Furthermore, dependency on EBI2 for
lymphocyte entry into LNs increased in a viral infection setting. We hypothesize this is due to elevated 7,25HC
levels and reduced chemokine levels in inflamed LNs. From these results, we aim to determine the step of LN
entry that EBI2 directs via a combination of in vivo homing assays, intravital imaging, and in vitro migration
assays (Aim 1). In addition, we will examine EBI2’s role in maintaining lymphocyte homing in inflamed LNs by
characterizing the changes, and signals mediating these changes, in LN oxysterol and chemokine levels during
infection (Aim 2). Overall, this work will define a novel chemoattractant driving lymphocyte migration into LNs
and advance our understanding of the transmigration process. As well as their role in LNs, HEVs form in tumor
associated tertiary lymphoid tissues and at sites of chronic inflammation. Our findings could inform development
of novel therapeutics that modulate lymphocyte infiltration in these disease settings.

Grant Number: 5F30AI176727-04
NIH Institute/Center: NIH
Principal Investigator: KEVIN CHEN